ABSTRACT Neuroinvasive flaviviruses such as West Nile virus (WNV), Japanese encephalitis virus (JEV), and Powassan virus (POWV) are transmitted by mosquitoes and ticks and are important causes of encephalitis in the US and worldwide. Typically only ~20% of individuals infected with flaviviruses exhibit symptoms, with a subset of these progressing to neuroinvasive disease. Polymorphisms in immune response genes, including OAS1 and CCR5 have been associated with WNV neuroinvasive disease in humans, but the factors that determine the pathologic outcome of flavivirus infection remain incompletely understood. We propose to use Collaborative Cross mice to identify immune response genes that mediate differential outcomes from neuroinvasive flavivirus infection. Mice provide an excellent model system for studying flavivirus neuroinvasive disease and flavivirus resistance is one of the earliest examples of a genetic determinant of pathogen susceptibility defined in mice. Over 80 years ago, flavivirus resistance was shown to be inherited in a single gene autosomal dominant manner and more than 15 years ago the trait was mapped to the Oas1b gene. Conventional laboratory mouse lines, including C57BL/6, have non-functional Oas1b alleles, whereas wild-derived lines encode full-length functional Oas1b. We used CRISPR/Cas-9 gene editing to generate two independent Oas1b-/- lines on the background of the Collaborative Cross line CC019 (which has a WSB-derived functional Oas1b allele). We found that CC019-Oas1b-/- mice were highly susceptible to WNV, JEV, and POWV, exhibiting 50-90% lethality compared to no lethality in parental CC019 mice. Although Oas1b had a large effect on flavivirus susceptibility, C57BL/6 mice were even more susceptible than CC019-Oas1b-/- mice, implying that non-Oas1b host factors contribute to the pathologic outcome of flavivirus infection in mice. To identify novel host immune factors that control pathogenesis of neuroinvasive flaviviruses generally, as well as factors that differentially restrict mosquito-borne (WNV, JEV) and tick-borne (POWV) viruses, we will assess JEV susceptibility in a panel of Collaborative Cross mice. Since we expect the well-described effect of Oas1b to dominate, we will test only the 47 (out of 69) Collaborative Cross lines with non-functional Oas1b alleles. We will use an F2 cross to map QTL associated with JEV susceptibility, viral load in the central nervous system, and the number and activation state of infiltrating leukocytes in the brain. We also will determine whether Collaborative Cross lines exhibit similar susceptibility to WNV, JEV, and POWV, or if there are differences between mosquito-borne and tick-borne flaviviruses. These studies will significantly advance our understanding of flavivirus neuroinvasive disease through the identification of polymorphic host genes associated with susceptibility to viral and immune-mediated neuropathology. This work will provide the foundation for future investigations of novel immune factors that control JEV pathogenesis, as well as their effects on other flaviviruses.